Granulocytic progenitor cells become progressively less capable of proliferation and survival with terminal differentiation. Granulocyte colony-stimulating factor (G-CSF) is the major regulator of granulopoiesis and stimulates the activation of multiple signaling pathways including the signal transducer and activator of transcription 5 (Stat5) pathway. Little is known about the activation status of G-CSF-stimulated signaling pathways at the distinct stages of granulocytic differentiation. When myeloid 32D cells transfected with the G-CSF receptor were induced to differentiate with G-CSF, Stat5 activation in response to G-CSF was gradually attenuated. Activation of other signaling molecules including Stat1, Stat3, Erk1/2, JNK and p38 was not altered significantly. Stat5 activation was also downregulated in multipotent FDCP-mix cells, which differentiated into mature granulocytes upon induction with G-CSF, but not in pro-B BaF/3 transfected with the G-CSF receptor, which showed no terminal granulocytic differentiation in response to G-CSF, suggesting that the effect of G-CSF is cell type specific. Attenuated activation of Stat5 correlated with reduced Stat5 protein levels, which was associated with expression of a protease activity capable of degrading Stat5 protein in vitro. The Stat5 protease activity was upregulated when myeloid cells were induced to differentiate with G-CSF, but its upregulation by G-CSF was blocked upon expression of leukemogenic proteins Bcr-Abl and Tel-Jak2. The activity of the Stat5 protease was inhibited partially by PMSF and completely by a1-antitrypsin, suggesting that it belongs to the serine family of protease. Our data provide the first evidence that a Stat5 protease activity is upregulated by G-CSF and may have important implications for understanding the molecular mechanism by which G-CSF orchestrates granulopoiesis.

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